US 2025/0019732 A1 · METHOD FOR CHEMICAL-BIOLOGICAL CASCADE SYNTHESIS OF L-PHOSPHINOTHRICIN AND MUTANTS THEREFOR

Abstract

A method for chemical-biological cascade synthesis of L-phosphinothricin is carried out as follows: 3-(methylethoxyphosphinyl) ethyl propionate is synthesized by addition reaction from diethoxymethylphosphine and acrylic acid, then a condensation reaction is carried out with 3-(methylethoxyphosphinyl) ethyl propionate and sodium ethoxide as reactants, then the product is subjected to a hydrolysis reaction with diethyl oxalate to synthesize 4-(hydroxymethylphosphinyl)-2-oxobutyric acid, and finally, L-phosphinothricin is catalytically synthesized by taking 4-(hydroxymethylphosphinyl)-2-oxobutyric acid as a raw material, and using highly active and stable wet cells co-expressing phsophinothricin dehydrogenase and alcohol dehydrogenase or co-expressing a phsophinothricin dehydrogenase mutant and alcohol dehydrogenase as a biocatalyst, thereby solving the problems of existing L-phosphinothricin synthesis being tedious, low asymmetric amination reduction activity and poor stability.

Claims11

Dependent claims

A method for synthesis of L-phosphinothricin, comprising the following steps:

forming a reaction system by using the wet cells obtained by fermentative cultivation of an engineered strain of recombinant Escherichia coli that co-express the gene encoding phosphinothricin dehydrogenase and the gene encoding alcohol dehydrogenase, or that co-express the gene encoding a phosphinothricin dehydrogenase mutant and the gene encoding alcohol dehydrogenase as a catalyst, using 4-(hydroxymethylphosphinyl)-2-oxobutyric acid as a substrate, adding isopropanol and NAD+, and using a buffer solution with a pH of 6-8 as the reaction medium, conducting the conversion reaction at 40-60° C. and 100-200 rpm, and subjecting the reaction solution to separation and purification to obtain L-phosphinothricin;
wherein the phosphinothricin dehydrogenase mutant is obtained by single or double mutation at position 73 or 91 of the phosphinothricin dehydrogenase with the amino acid sequence shown as SEQ ID NO.2.

Dependencies are inferred from the claim text.

Method for chemical-biological cascade synthesis of l-phosphinothricin and mutants therefor

Life of this patent

Method for chemical-biological cascade synthesis of l-phosphinothricin and mutants therefor

Life of this patent

Abstract

Claims11